Particles for inhalation having sustained release properties

ABSTRACT

The invention generally relates to a method for pulmonary delivery of therapeutic, prophylactic and diagnostic agents to a patient wherein the agent is released in a sustained fashion, and to particles suitable for use in the method. In particular, the invention relates to a method for the pulmonary delivery of a therapeutic, prophylactic or diagnostic agent comprising administering to the respiratory tract of a patient in need of treatment, prophylaxis or diagnosis an effective amount of particles comprising a therapeutic, prophylactic or diagnostic agent or any combination thereof in association with a charged lipid, wherein the charged lipid has an overall net charge which is opposite to that of the agent upon association with the agent. Release of the agent from the administered particles occurs in a sustained fashion.

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.09/752,106, filed Dec. 29, 2000, which is a continuation-in-part ofapplication Ser. No. 09/394,233, filed Sep. 13, 1999, which is acontinuation-in-part of application Ser. No. 08/971,791, filed Nov. 17,1997, now U.S. Pat. No. 5,985,309, which claims the benefit of U.S.Provisional Application No. 60/059,004, filed Sep. 15, 1997.

[0002] This application is also related to application Ser. No.09/337,245, filed Jun. 22, 1999; Ser. No. 09/383,054, filed on Aug. 25,1999; Ser. No. 09/382,959, filed Aug. 25, 1999; Ser. No. 09/644,320,filed on Aug. 23, 2000; Ser. No. 09/665,252, filed Sep. 19, 2000, nowU.S. Pat. No. 6,514,482; 09/644,105, filed Aug. 23, 2000; Ser. No.09/644,736, filed Aug. 23, 2000; and Ser. No. 09/591,307, filed Jun. 9,2000. The entire teachings of the above applications are incorporatedherein by reference.

GOVERNMENT SUPPORT

[0003] This invention was made with government support under NIH GrantNumber HD29129 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0004] Pulmonary delivery of bioactive agents, for example, therapeutic,diagnostic and and prophylactic agents provides an attractivealternative to, for example, oral, transdermal and parenteraladministration. That is, pulmonary administration can typically becompleted without the need for medical intervention(self-administration), the pain often associated with injection therapyis avoided, and the amount of enzymatic and pH mediated degradation ofthe bioactive agent, frequently encountered with oral therapies, can besignificantly reduced. In addition, the lungs provide a large mucosalsurface for drug absorption and there is no first-pass liver effect ofabsorbed drugs. Further, it has been shown that high bioavailability ofmany molecules, for example, macromolecules, can be achieved viapulmonary delivery or inhalation. Typically, the deep lung, or alveoli,is the primary target of inhaled bioactive agents, particularly foragents requiring systemic delivery.

[0005] The release kinetics or release profile of a bioactive agent intothe local and/or systemic circulation is a key consideration in mosttherapies, including those employing pulmonary delivery. That is, manyillnesses or conditions require administration of a constant orsustained levels of a bioactive agent to provide an effective therapy.Typically, this can be accomplished through a multiple dosing regimen orby employing a system that releases the medicament in a sustainedfashion.

[0006] However, delivery of bioactive agents to the pulmonary systemtypically results in rapid release of the agent followingadministration. For example, U.S. Pat. No. 5,997,848 to Patton et al.describes the rapid absorption of insulin following administration of adry powder formulation via pulmonary delivery. The peak insulin levelwas reached in about 30 minutes for primates and in about 20 minutes forhuman subjects. Further, Heinemann, Traut and Heise teach in DiabeticMedicine 14:63-72 (1997) that the onset of action, assessed by glucoseinfusion rate, in healthy volunteers after inhalation was rapid with thehalf-maximal action reached in about 30 minutes.

[0007] As such, a need exists for formulations suitable for inhalationcomprising bioactive agents and wherein the bioactive agent of theformulation is released in a sustained fashion into the systemic and/orlocal circulation.

SUMMARY OF THE INVENTION

[0008] This invention is based upon the unexpected discovery thatcombining a charged agent with a lipid carrying an opposite chargeresults in a sustained release profile of the agent.

[0009] The invention generally relates to a method for pulmonarydelivery of therapeutic, prophylactic and diagnostic agents to a patientwherein the agent is released in a sustained fashion, and to particlessuitable for use in the method. In particular, the invention relates toa method for the pulmonary delivery of a therapeutic, prophylactic ordiagnostic agent comprising administering to the respiratory tract of apatient in need of treatment, prophylaxis or diagnosis an effectiveamount of particles comprising a therapeutic, prophylactic or diagnosticagent or any combination thereof in association with a charged lipid,wherein the charged lipid has an overall net charge which is opposite tothat of the agent upon association with the agent. Release of the agentfrom the administered particles occurs in a sustained fashion.

[0010] In one embodiment, the association of the therapeutic,prophylactic or diagnostic agent and the oppositely charged lipid canresult from ionic complexation. In another embodiment, association ofthe therapeutic, prophylactic or diagnostic agent and the oppositelycharged lipid can result from hydrogen bonding.

[0011] In yet a further embodiment, the association of the therapeutic,prophylactic or diagnostic agent and the oppositely charged lipid canresult from a combination of ionic complexation and hydrogen bonding.

[0012] The particles suitable for use in the method can comprise atherapeutic, prophylactic or diagnostic agent in association with acharged lipid having a charge opposite to that of the agent. The chargesare opposite upon association, prior to administration. In a preferredembodiment, the charges of the agent and lipid upon association, priorto administration, are those which the agent and lipid possess atpulmonary pH.

[0013] For example, the particles suitable for pulmonary delivery cancomprise a therapeutic, prophylactic or diagnostic agent which possessesan overall net negative charge, in association with a lipid whichpossesses an overall net positive charge. For example, the agent can beinsulin which has an overall net charge which is negative and the lipidcan be 1,2-dipalmitoyl-sn-glycero-3-ethylphosphatidylcholine (DPePC).

[0014] Alternatively, the particles suitable for pulmonary delivery cancomprise a therapeutic, prophylactic or diagnostic agent which possessesan overall net positive charge in association with a lipid whichpossesses an overall net negative charge. For example, the agent can bealbuterol which possesses an overall positive charge and the lipid canbe 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)](DPPG) whichpossesses an overall net negative charge.

[0015] Further, the particles suitable for pulmonary delivery cancomprise a therapeutic, prophylactic or diagnostic agent which has anoverall net charge which can be modified by adjusting the pH of asolution of the agent, prior to association with the lipid. For example,at a pH of about 7.4 insulin has an overall net charge which isnegative. Therefore, insulin and a positively charged lipid can beassociated at this pH prior to administration to prepare a particlehaving an agent in association with a charged lipid wherein the chargedlipid has a charge opposite to that of the agent. However, the chargeson insulin can also be modified, when in solution, to possess an overallnet charge which is positive by modifying the pH of the solution to beless than the pI of insulin (pI=5.5). As such, when insulin is insolution at a pH of about 4, for example, it will possess an overall netcharge which is positive. As this is the case, the positively chargedinsulin can be associated with a negatively charged lipid, for example,1,2-distearoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DSPG).

[0016] Modification of the charge of the therapeutic, prophylactic ordiagnostic agent prior to association with the charged lipid, can beaccomplished with many agents, particularly, proteins. For example,charges on proteins can be modulated by spray drying feed solutionsbelow or above the isoelectric points (pI) of the protein. Chargemodulation can also be accomplished for small molecules by spray dryingfeed solutions below or above the pKa of the molecule.

[0017] In a particular embodiment, the particles of the inventioncomprise more than one lipid, more than one bioactive agent or both.Also charged lipids can be combined with lipids without a net charge.

[0018] The particles, can further comprise a carboxylic acid which isdistinct from the bioactive agent and lipid. In one embodiment, thecarboxylic acid includes at least two carboxyl groups. Carboxylic acids,include the salts thereof as well as combinations of two or morecarboxylic acids and/or salts thereof. In a preferred embodiment, thecarboxylic acid is a hydrophilic carboxylic acid or salt thereof. Citricacid and citrates, such as, for example sodium citrate, are preferred.Combinations or mixtures of carboxylic acids and/or their salts also canbe employed.

[0019] The particles suitable for use in the invention can furthercomprise a multivalent salt or its ionic components. In a preferredembodiment, the salt is a divalent salt. In another preferredembodiment, the salt is a salt of an alkaline-earth metal, such as, forexample, calcium chloride. The particles of the invention can alsoinclude mixtures or combinations of salts and/or their ionic components.

[0020] The particles suitable for use in the invention can furthercomprise an amino acid. In a preferred embodiment the amino acid ishydrophobic.

[0021] The particles, also referred to herein as powder, can be in theform of a dry powder suitable for inhalation. The particles can have atap density of less than about 0.4 g/cm³, preferably less than about 0.1g/cm³. Further, the particles suitable for use in the invention can havea median geometric diameter of from about 5 micrometers to about 30micrometers. In yet another embodiment, the particles suitable for usein the invention have an aerodynamic diameter of from about 1 to about 5micrometers.

[0022] The invention has numerous advantages. For example, particlessuitable for inhalation can be designed to possess a sustained releaseprofile. This sustained released profile provides for prolongedresidence of the administered bioactive agent in the lung and increasesthe amount of time in which therapeutic levels of the agent are presentin the local environment or systemic circulation. The sustained releaseof agent provides a desirable alternative to injection therapy currentlyused for many therapeutic, diagnostic and prophylactic agent requiringsustained release of agent, such as insulin for the treatment ofdiabetes. In addition, the invention provides a method of delivery tothe pulmonary system wherein the high initial release of agent typicallyseen in inhalation therapy is reduced. Consequently, patient complianceand comfort can be increased by not only reducing frequency of dosing,but by providing a therapy which is more amenable to patients.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 is a graph showing the in vivo release profile of drypowder formulations comprising insulin and either a lipid (DPPC) with nooverall net a charge or lipid having an overall net charge opposite tothat of insulin (DSePC and DPePC).

[0024]FIG. 2 is a graph of the in vivo release profile of dry powderformulations comprising insulin in combination with a lipid with nooverall net charge (DPPC) or insulin in combination with a charged lipidhaving an overall negative charge (DPPG) and spray dried with the activeagent at either pH 4 or 7.4.

[0025]FIG. 3 is a graph showing the in vivo release profile of drypowder formulation comprising estrone sulfate (−) and either a lipidwith no overall net charge (DPPC) or a lipid having an overall chargeopposite (DPePC, +) to that of the estrone sulfate.

[0026]FIG. 4 is a graph of percent of PenH above baseline versus timefollowing administration of dry powder formulations of albuterol sulfateand lipid in animals which have been challenged repeatedly over timewith methacholine given by nebulization.

[0027] The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings. The drawings are not necessarily to scale,emphasis instead being placed upon illustrating the principles of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

[0028] A description of preferred embodiments of the invention follows.

[0029] Therapeutic, prophylactic or diagnostic agents, can also bereferred to herein as “bioactive agents,” “medicaments” or “drugs.”

[0030] The invention relates to a method for the pulmonary delivery oftherapeutic, prophylactic and diagnostic agents comprising administeringto the respiratory tract of a patient in need of treatment, prophylaxisor diagnosis an effective amount of particles comprising a therapeutic,prophylactic or diagnostic agent or any combination thereof inassociation with a charged lipid, wherein the charged lipid has anoverall net charge which is opposite to that of the agent. The agent isreleased from the administered particles in a sustained fashion.

[0031] The particles of the invention release bioactive agent in asustained fashion. As such, the particles possess sustained releaseproperties. “Sustained release”, as that term is used herein, refers toa release of active agent in which the period of release of an effectivelevel of agent is longer than that seen with the same bioactive agentwhich is not associated with an oppositely charged lipid, prior toadministration. In addition, a sustained release also refers to areduction in the burst of agent typically seen in first two hoursfollowing administration, and more preferably in the first hour, oftenreferred to as the initial burst. In a preferred embodiment, thesustained release is characterized by both the period of release beinglonger in addition to a decreased burst. For example, a sustainedrelease of insulin can be a release showing elevated levels out to atleast 4 hours post administration, such as about 6 hours or more.

[0032] “Pulmonary delivery,” as that term is used herein refers todelivery to the respiratory tract. The “respiratory tract,” as definedherein, encompasses the upper airways, including the oropharynx andlarynx, followed by the lower airways, which include the tracheafollowed by bifurcations into the bronchi and bronchioli (e.g., terminaland respiratory). The upper and lower airways are called the conductingairways. The terminal bronchioli then divide into respiratory bronchioliwhich then lead to the ultimate respiratory zone, namely, the alveoli,or deep lung. The deep lung, or alveoli, are typically the desired thetarget of inhaled therapeutic formulations for systemic drug delivery.

[0033] In one embodiment, the therapeutic, prophylactic or diagnosticagent and the oppositely charged lipid can be in association primarilyas a result of ionic bonding, for example, ionic complexation. Inanother embodiment, the therapeutic, prophylactic or diagnostic agentand the oppositely charged lipid can be in association primarily as aresult of hydrogen bonding. It is understood that a combination of ionicand hydrogen bonding can contribute to the association of the bioactiveand charged lipid.

[0034] Ionic bonding is bonding which occurs via charge/chargeinteractions between atoms or groups of atoms. Since opposite chargesattract, the atoms in an ionic compound are held together by thisattraction.

[0035] Hydrogen bonding refers to bonding wherein a hydrogen atom isshared between two molecules. For example, a hydrogen atom covalentlyattached to an electronegative atom such as nitrogen, oxygen, sulfur orphosphorous shares its partial positive charge with a secondelectronegative atom, for example, nitrogen, oxygen, sulfur orphosphorous.

[0036] The particles suitable for use in the method can comprise atherapeutic, prophylactic or diagnostic agent in association with acharged lipid having a charge opposite to that of the agent uponassociation, prior to administration. In a preferred embodiment, thecharges possessed by the agent and lipid, upon association, are the sameas the charges which the agent and lipid possess at pulmonary pHfollowing administration.

[0037] For example, the particles suitable for pulmonary delivery cancomprise a therapeutic, prophylactic or diagnostic agent which possessesan overall net negative charge in association with a lipid whichpossesses an overall net positive charge. For example, the agent can beinsulin and the lipid can be an alkylphosphatidylcholine, such as1,2-dipalmitoyl-sn-glycero-3-ethylphosphatidylcholine (DPePC).

[0038] Alternatively, the particles suitable for pulmonary delivery cancomprise a therapeutic, prophylactic or diagnostic agent which possessesan overall net positive charge in association with a lipid whichpossesses an overall net negative charge, preferably in the pulmonary pHrange. For example, the agent can be albuterol sulfate which possessesan overall positive charge and the lipid can be1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG) whichpossesses an overall net negative charge.

[0039] Further, the particles suitable for pulmonary delivery cancomprise a therapeutic, prophylactic or diagnostic agent which has anoverall net charge which can be modified by adjusting the pH of asolution of the agent prior to association with the charged lipid. Forexample, at a pH of about 7.4 insulin has an overall net charge which isnegative. Therefore, insulin and a positively charged lipid can beassociated at this pH, prior to administration, to prepare a particlehaving an bioactive agent in association with a charged lipid whereinthe charged lipid has a charge opposite to that of the agent uponassociation. However, insulin can also be modified when in solution topossess an overall net charge which is positive by modifying the pH ofthe solution to be less than the pI of insulin (pI=5.5). As such, wheninsulin is in solution at a pH of 4, for example, it will possess anoverall net charge which is positive. As this is the case, thepositively charged insulin can be associated with a negatively chargedlipid, for example,1,2-distearoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DSPG).Modification of the charge of the therapeutic, prophylactic ordiagnostic agent is applicable to many agents, particularly, proteins.

[0040] “Pulmonary pH range”, as that term is used herein, refers to thepH range which can be encountered in the lung of a patient. Typically,in humans, this range of pH is from about 6.4 to about 7.0, such as from6.4 to about 6.7. pH values of the airway lining fluid (ALF) have beenreported in “Comparative Biology of the Normal Lung”, CRC Press, (1991)by R. A. Parent and range from 6.44 to 6.74)

[0041] “Charged lipid” as that term is used herein, refers to lipidswhich are capable of possessing an overall net charge. The charge on thelipid can be negative or positive. The lipid can be chosen to have acharge opposite to that of the active agent when the lipid and activeagent are associated. In a preferred embodiment the charged lipid is acharged phospholipid. Preferably, the phospholipid is endogenous to thelung or can be metabolized upon administration to a lung endogenousphospholipid. Combinations of charged lipids can be used. Thecombination of charged lipid also has an overall net charge opposite tothat of the bioactive agent upon association.

[0042] The charged phospholipid can be a negatively charged lipid suchas, a 1,2-diacyl-sn-glycero-3-[phospho-rac-(1-glycerol)] and a1,2-diacyl-sn-glycerol-3-phosphate.

[0043] The 1,2-diacyl-sn-glycero-3-[phospho-rac-(1-glycerol)]phospholipids can be represented by the Formula I:

[0044] wherein R₁ and R₂ are independently aliphatic groups having fromabout 3 to about 24 carbon atoms, preferably from about 10 to about 20carbon atoms.

[0045] Aliphatic group as that term is used herein in Formulas I-VIrefers to substituted or unsubstituted straight chained, branched orcyclic C₁-C₂₄ hydrocarbons which can be completely saturated, which cancontain one or more heteroatoms such as nitrogen, oxygen or sulfurand/or which can contain one or more units of unsaturation.

[0046] Suitable substituents on an aliphatic group include —OH, halogen(—Br, —Cl, —I and —F) —O(aliphatic, substituted), —CN, —NO₂, —COOH,—NH₂, —NH(aliphatic group, substituted aliphatic), —N(aliphatic group,substituted aliphatic group)₂, —COO(aliphatic group, substitutedaliphatic group), —CONH₂, —CONH(aliphatic, substituted aliphatic group),—SH, —S(aliphatic, substituted aliphatic group) and —NH—C(═NH)—NH₂. Asubstituted aliphatic group can also have a benzyl, substituted benzyl,aryl (e.g., phenyl, naphthyl or pyridyl) or substituted aryl group as asubstituent. A substituted aliphatic can have one or more substituents.

[0047] Specific examples of this type of negatively charged phospholipidinclude, but are not limited to,1,2-distearoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DSPG),1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG),1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol)] (DPPG),1,2-dilauroyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DLPG), and1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG).

[0048] The 1,2-diacyl-sn-glycerol-3-phosphate phospholipids can berepresented by the Formula II:

[0049] R₁ and R₂ are independently an aliphatic group having from about3 to about 24 carbon atoms, preferably from about 10 to about 20 carbonatoms.

[0050] Specific examples of this type of phospholipid include, but arenot limited to, 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA),1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA),1,2-dioleoyl-sn-glycero-3-phosphate (DOPA),1,2-distearoyl-sn-glycero-3-phosphate (DSPA), and1,2-dilauroyl-sn-glycero-3-phosphate (DLPA).

[0051] The charged lipid can be a positively charged lipid such as a1,2-diacyl-sn-glycero-3-alkylphosphocholine and a1,2-diacyl-sn-glycero-3-alkylphosphoalkanolamine.

[0052] The 1,2-diacyl-sn-glycero-3-alkyllphosphocholine phospholipidscan be represented by the Formula III:

[0053] wherein R₁ and R₂ are independently an aliphatic group havingfrom about 3 to about 24 carbon atoms, preferably from about 10 to about20 carbon atoms. R₃ is an aliphatic group having from about 1 to about24 carbons, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl,heptyl, octyl and the like.

[0054] Specific examples of this type of positively charged phospholipidinclude, but are not limited to,1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (DPePC),1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMePC),1,2-distearoyl-sn-glycero-3 -ethylphosphocholine (DSePC),1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLePC), and1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOePC). The1,2-diacyl-sn-glycero-3-alkylphosphoalkanolamine phospholipids can berepresented by the Formula IV:

[0055] wherein R₁ and R₂ are independently an aliphatic group havingfrom about 3 to about 24 carbon atoms, preferably from about 10 to about20 carbon atoms. R₃ is an aliphatic group having from about 1 to about24 carbons, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl,heptyl, octyl and the like. R₄ is independently hydrogen, or analiphatic group having from about 1 to about 6 carbon atoms.

[0056] Specific examples of this type of positively charged phospholipidinclude, but are not limited to,1,2-dipalmitoyl-sn-glycero-3-ethylethanolamine (DPePE),1,2-dimyristoyl-sn-glycero-3-ethylphosphoethanolamine (DMePE),1,2-distearoyl-sn-glycero-3-ethylphosphoethanolamine (DSePE),1,2-dilauroyl-sn-glycero-3-ethylphosphoethanolamine (DLePE), and1,2-dioleoyl-sn-glycero-3-ethylphosphoethanolamine (DOePE).

[0057] Other charged lipids suitable for use in the invention includethose described in U.S. Pat. No. 5,466,841 to Horrobin et al. issued onNov. 14, 1995, U.S. Pat. Nos. 5,698,721 and 5,902,802 to Heath issuedDec. 16, 1997 and May 11, 1999, respectively, and U.S. Pat. No.4,480,041 to Myles et al. issued Oct. 30, 1984, the entire contents ofall of which are incorporated herein by reference.

[0058] The charged lipid and the therapeutic, prophylactic or diagnosticagent can be present in the particles of the invention at a charge ratioof lipid to active of from about 0.25:1 or more, preferably from about0.25:1 to about 1:0.25, for example, about 0.5:1 to about 1:0.5.Preferably the charge ratio is about 1:1. When an excess of charge ispresent, it is preferred that the excess charge is contributed by thelipid.

[0059] A suitable charge ratio can be determined as follows. First, thenumber of charges present on both the bioactive agent and lipid, at theconditions under which association of the two will occur, prior toadministration, should be determined. Next, the equivalent weight ofboth the bioactive agent and lipid should be determined. This can becarried out following the example below employing insulin as thebioactive agent and DPePC as the charged lipid at a pH of about 7.4.Molecular Weight of Insulin: 5,800 g/mole Number of Negative Charges onInsulin: 6 equivalent Equivalent Weight Per Charge: 5,800 × 1/6 = 967 gMolecular Weight of DPePC:   763 g/mole Number of Negative Charges onDPePC: 1 equivalent Equivalent Weight Per Charge: 763 × 1/1 = 763 g

[0060] Therefore, to obtain for example, a 1:1 charge ratio of DPePC toinsulin

[0061] 763 g DPePC is associated with 967 g insulin

[0062] OR

[0063] 1 g DPePC is associated with 1.27 (967/763=1.27) g insulin.

[0064] Alternatively,

[0065] 967 g insulin is associated with 763 g DPePC

[0066] OR

[0067] 1 g insulin is associated with 0.79 (763/967=0.79) g DPePC.

[0068] In molar terms,

[0069] 1 mole DPePC is associated with ⅙ mole insulin

[0070] OR

[0071] 1 mole insulin is associated with 6 moles DPePC.

[0072] This analysis can be used to determine the amount of lipid andactive agent needed for any ratio desired and any combination ofbioactive agent and lipid.

[0073] The charged lipid can be present in the particles in an amountranging from about 1 to about 99% by weight. Preferably, the chargedlipid is present in the particles in an amount ranging from about 10% toabout 90% by weight.

[0074] The particles of the invention can also comprise phospholipids,which are zwitterionic and therefore do not possess an overall netcharge. Such lipids, can assist in providing particles with the propercharacterisitics for inhalation. Such phospholipids suitable for use inthe invention include, but are not limited to, a1,2-diacyl-sn-glycero-3-phosphocholine and a1,2-diacyl-sn-glycero-3-phosphoalkanolamine. These lipids can preferablybe present in the particles in an amount ranging from about 10% to about90% by weight. Preferably, these lipids can be present in the particlesin an amount ranging from abut 50% to about 80% by weight.

[0075] The 1,2-diacyl-sn-glycero-3-phosphocholine phospholipids can berepresented by Formula V:

[0076] R₁ and R₂ are independently an aliphatic group having from about3 to about 24 carbon atoms, preferably from about 10 to about 20 carbonatoms. R₄ is independently hydrogen, or an aliphatic group having fromabout 1 to about 6 carbon atoms.

[0077] Specific examples of 1,2-diacyl-sn-glycero-3-phosphocholinephospholipids include, but are not limited to,1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dilaureoyl-sn-3-glycero-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),

[0078] The 1,2-diacyl-sn-glycero-3-phosphoalkanolamine phospholipids canbe represented by Formula VI:

[0079] wherein R₁ and R₂ are independently an aliphatic group havingfrom about 3 to about 24 carbon atoms, preferably, from about 10 toabout 20 carbon atoms and R₄ is independently hydrogen or an aliphaticgroup having from about 1 to about 6 carbon atoms.

[0080] Specific examples of this type of phospholipid include, but arenot limited to, 1,2-dipalmitoyl-sn-glycero-3-ethanolamine (DPPE),1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), and1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0081] Therapeutic, prophylactic or diagnostic agents, can also bereferred to herein as “bioactive agents,” “medicaments” or “drugs.” Itis understood that one or more bioactive agents can be present in theparticles of the invention. Hydrophilic as well as hydrophobic agentscan be used. The agent must be capable of possessing an overall netcharge. The amount of bioactive agent present in the particles of theinvention can be from about 0.1 weight % to about 95 weight %, forexample, from about 5 to about 75%, such as from about 10 to about 50%.Particles in which the drug is distributed throughout a particle arepreferred.

[0082] Suitable bioactive agents include agents which can act locally,systemically or a combination thereof. The term “bioactive agent,” asused herein, is an agent, or its pharmaceutically acceptable salt, whichwhen released in vivo, possesses the desired biological activity, forexample therapeutic, diagnostic and/or prophylactic properties in vivo.

[0083] Examples of bioactive agent include, but are not limited to,synthetic inorganic and organic compounds, proteins and peptides,polysaccharides and other sugars, lipids, and DNA and RNA nucleic acidsequences having therapeutic, prophylactic or diagnostic activities.Agents with a wide range of molecular weight can be used, for example,between 100 and 500,000 grams or more per mole.

[0084] The agents can have a variety of biological activities, such asvasoactive agents, neuroactive agents, hormones, anticoagulants,immunomodulating agents, cytotoxic agents, prophylactic agents,antibiotics, antivirals, antisense, antigens, antineoplastic agents andantibodies.

[0085] Proteins, include complete proteins, muteins and active fragmentsthereof, such as insulin, immunoglobulins, antibodies, cytokines (e.g.,lymphokines, monokines, chemokines), interleukins, interferons (β-IFN,α-IFN and γ-IFN), erythropoietin, nucleases, tumor necrosis factor,colony stimulating factors, enzymes (e.g. superoxide dismutase, tissueplasminogen activator), tumor suppressors, blood proteins, hormones andhormone analogs (e.g., growth hormone, adrenocorticotropic hormone andluteinizing hormone releasing hormone (LHRH)), vaccines (e.g., tumoral,bacterial and viral antigens), antigens, blood coagulation factors;growth factors; granulocyte colony-stimulating factor (“G-CSF”);peptides include protein inhibitors, protein antagonists, and proteinagonists, calcitonin; nucleic acids include, for example, antisensemolecules, oligonucleotides, and ribozymes. Polysaccharides, such asheparin, can also be administered.

[0086] Bioactive agent for local delivery within the lung, include suchas agents as those for the treatment of asthma, chronic obstructivepulmonary disease (COPD), emphysema, or cystic fibrosis. For example,genes for the treatment of diseases such as cystic fibrosis can beadministered, as can beta agonists steroids, anticholinergics, andleukotriene modifers for asthma.

[0087] Other specific bioactive agents include, estrone sulfate,albuterol sulfate, parathyroid hormone-related peptide, somatostatin,nicotine, clonidine, salicylate, cromolyn sodium, salmeterol,formeterol, L-dopa, Carbidopa or a combination thereof, gabapenatin,clorazepate, carbamazepine and diazepam.

[0088] Nucleic acid sequences include genes, antisense molecules whichcan, for instance, bind to complementary DNA to inhibit transcription,and ribozymes.

[0089] The particles can include any of a variety of diagnostic agentsto locally or systemically deliver the agents following administrationto a patient. For example, imaging agents which include commerciallyavailable agents used in positron emission tomography (PET), computerassisted tomography (CAT), single photon emission computerizedtomography, x-ray, fluoroscopy, and magnetic resonance imaging (MRI) canbe employed.

[0090] Examples of suitable materials for use as contrast agents in MRIinclude the gadolinium chelates currently available, such as diethylenetriamine pentacetic acid (DTPA) and gadopentotate dimeglumine, as wellas iron, magnesium, manganese, copper and chromium.

[0091] Examples of materials useful for CAT and x-rays include iodinebased materials for intravenous administration, such as ionic monomerstypified by diatrizoate and iothalamate and ionic dimers, for example,ioxagalte.

[0092] Diagnostic agents can be detected using standard techniquesavailable in the art and commercially available equipment.

[0093] The particles can further comprise a carboxylic acid which isdistinct from the agent and lipid. In one embodiment, the carboxylicacid includes at least two carboxyl groups. Carboxylic acids, includethe salts thereof as well as combinations of two or more carboxylicacids and/or salts thereof. In a preferred embodiment, the carboxylicacid is a hydrophilic carboxylic acid or salt thereof. Suitablecarboxylic acids include but are not limited to hydroxydicarboxylicacids, hydroxytricarboxilic acids and the like. Citric acid andcitrates, such as, for example sodium citrate, are preferred.Combinations or mixtures of carboxylic acids and/or their salts also canbe employed.

[0094] The carboxylic acid can be present in the particles in an amountranging from about 0 to about 80% weight. Preferably, the carboxylicacid can be present in the particles in an amount of about 10 to about20%.

[0095] The particles suitable for use in the invention can furthercomprise a multivalent salt or its ionic components. As used herein, a“multivalent” salt refers to salts having a ionic component with avalency greater than one. For example, divalent salts. In a preferredembodiment, the salt is a divalent salt. In another preferredembodiment, the salt is a salt of an alkaline-earth metal, such as, forexample, calcium chloride. The particles of the invention can alsoinclude mixtures or combinations of salts and/or their ionic components.

[0096] The salt or its ionic components are present in the particles inan amount ranging from about 0 to about 40% weight.

[0097] The particles suitable for use in the invention can furthercomprise an amino acid. In a preferred embodiment the amino acid ishydrophobic. Suitable naturally occurring hydrophobic amino acids,include but are not limited to, leucine, isoleucine, alanine, valine,phenylalanine, glycine and tryptophan. Combinations of hydrophobic aminoacids can also be employed Non-naturally occurring amino acids include,for example, beta-amino acids. Both D, L configurations and racemicmixtures of hydrophobic amino acids can be employed. Suitablehydrophobic amino acids can also include amino acid derivatives oranalogs. As used herein, an amino acid analog includes the D or Lconfiguration of an amino acid having the following formula:—NH—CHR—CO—, wherein R is an aliphatic group, a substituted aliphaticgroup, a benzyl group, a substituted benzyl group, an aromatic group ora substituted aromatic group and wherein R does not correspond to theside chain of a naturally-occurring amino acid. As used herein,aliphatic groups include straight chained, branched or cyclic C1-C8hydrocarbons which are completely saturated, which contain one or twoheteroatoms such as nitrogen, oxygen or sulfur and/or which contain oneor more units of unsaturation. Aromatic or aryl groups includecarbocyclic aromatic groups such as phenyl and naphthyl and heterocyclicaromatic groups such as imidazolyl, indolyl, thienyl, furanyl, pyridyl,pyranyl, oxazolyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyland acridintyl.

[0098] Suitable substituents on an aliphatic, aromatic or benzyl groupinclude —OH, halogen (u—Br, —Cl, —I and —F) —O(aliphatic, substitutedaliphatic, benzyl, substituted benzyl, aryl or substituted aryl group),—CN, —NO₂, —COOH, —NH₂, —NH(aliphatic group, substituted aliphatic,benzyl, substituted benzyl, aryl or substituted aryl group),—N(aliphatic group, substituted aliphatic, benzyl, substituted benzyl,aryl or substituted aryl group)₂, —COO(aliphatic group, substitutedaliphatic, benzyl, substituted benzyl, aryl or substituted aryl group),—CONH₂, —CONH(aliphatic, substituted aliphatic group, benzyl,substituted benzyl, aryl or substituted aryl group)), —SH, —S(aliphatic,substituted aliphatic, benzyl, substituted benzyl, aromatic orsubstituted aromatic group) and —NH—C(═NH)—NH₂. A substituted benzylicor aromatic group can also have an aliphatic or substituted aliphaticgroup as a substituent. A substituted aliphatic group can also have abenzyl, substituted benzyl, aryl or substituted aryl group as asubstituent. A substituted aliphatic, substituted aromatic orsubstituted benzyl group can have one or more substituents. Modifying anamino acid substituent can increase, for example, the lypophilicity orhydrophobicity of natural amino acids which are hydrophilic.

[0099] A number of the suitable amino acids, amino acids analogs andsalts thereof can be obtained commercially. Others can be synthesized bymethods known in the art. Synthetic techniques are described, forexample, in Green and Wuts, “Protecting Groups in Organic Synthesis”,John Wiley and Sons, Chapters 5 and 7, 1991.

[0100] Hydrophobicity is generally defined with respect to the partitionof an amino acid between a nonpolar solvent and water. Hydrophobic aminoacids are those acids which show a preference for the nonpolar solvent.Relative hydrophobicity of amino acids can be expressed on ahydrophobicity scale on which glycine has the value 0.5. On such ascale, amino acids which have a preference for water have values below0.5 and those that have a preference for nonpolar solvents have a valueabove 0.5. As used herein, the term hydrophobic amino acid refers to anamino acid that, on the hydrophobicity scale has a value greater orequal to 0.5, in other words, has a tendency to partition in thenonpolar acid which is at least equal to that of glycine.

[0101] Examples of amino acids which can be employed include, but arenot limited to: glycine, proline, alanine, cysteine, methionine, valine,leucine, tyrosine, isoleucine, phenylalanine, tryptophan. Preferredhydrophobic amino acids include leucine, isoleucine, alanine, valine,phenylalanine, glycine and tryptophan. Combinations of hydrophobic aminoacids can also be employed. Furthermore, combinations of hydrophobic andhydrophilic (preferentially partitioning in water) amino acids, wherethe overall combination is hydrophobic, can also be employed.Combinations of one or more amino acids can also be employed.

[0102] The amino acid can be present in the particles of the inventionin an amount from about 0% to about 60 weight %. Preferably, the aminoacid can be present in the particles in an amount ranging from about 5to about 30 weight %. The salt of a hydrophobic amino acid can bepresent in the particles of the invention in an amount of from about 0%to about 60 weight %. Preferably, the amino acid salt is present in theparticles in an amount ranging from about 5 to about 30 weight %.Methods of forming and delivering particles which include an amino acidare described in U.S. patent application Ser. No. 09/382,959, filed onAug. 25, 1999, entitled Use of Simple Amino Acids to Form PorousParticles During Spray Drying the entire teaching of which isincorporated herein by reference.

[0103] In a further embodiment, the particles can also include othermaterials such as, for example, buffer salts, dextran, polysaccharides,lactose, trehalose, cyclodextrins, proteins, peptides, polypeptides,fatty acids, fatty acid esters, inorganic compounds, phosphates.

[0104] In one embodiment of the invention, the particles can furthercomprise polymers. The use of polymers can further prolong release.Biocompatible or biodegradable polymers are preferred. Such polymers aredescribed, for example, in U.S. Pat. No. 5,874,064, issued on Feb. 23,1999 to Edwards et al., the teachings of which are incorporated hereinby reference in their entirety.

[0105] In yet another embodiment, the particles include a surfactantother than one of the charged lipids described above. As used herein,the term “surfactant” refers to any agent which preferentially absorbsto an interface between two immiscible phases, such as the interfacebetween water and an organic polymer solution, a water/air interface ororganic solvent/air interface. Surfactants generally possess ahydrophilic moiety and a lipophilic moiety, such that, upon absorbing tomicroparticles, they tend to present moieties to the externalenvironment that do not attract similarly-coated particles, thusreducing particle agglomeration. Surfactants may also promote absorptionof a therapeutic or diagnostic agent and increase bioavailability of theagent.

[0106] Suitable surfactants which can be employed in fabricating theparticles of the invention include but are not limited to hexadecanol;fatty alcohols such as polyethylene glycol (PEG);polyoxyethylene-9-lauryl ether; a surface active fatty acid, such aspalmitic acid or oleic acid; glycocholate; surfactin; a poloxomer; asorbitan fatty acid ester such as sorbitan trioleate (Span 85); andtyloxapol.

[0107] The surfactant can be present in the particles in an amountranging from about 0 to about 60 weight %. Preferably, it can be presentin the particles in an amount ranging from about 5 to about 50 weight %.

[0108] It is understood that when the particles includes a carboxylicacid, a multivalent salt, an amino acid, a surfactant or any combinationthereof that interaction between these components of the particle andthe charged lipid can occur.

[0109] The particles, also referred to herein as powder, can be in theform of a dry powder suitable for inhalation. In a particularembodiment, the particles can have a tap density of less than about 0.4g/cm³. Particles which have a tap density of less than about 0.4 g/cm³are referred to herein as “aerodynamically light particles.” Morepreferred are particles having a tap density less than about 0.1 g/cm³.

[0110] Aerodynamically light particles have a preferred size, e.g., avolume median geometric diameter (VMGD) of at least about 5 microns(μm). In one embodiment, the VMGD is from about 5 μm to about 30 μm. Inanother embodiment of the invention, the particles have a VMGD rangingfrom about 9 μm to about 30 μm. In other embodiments, the particles havea median diameter, mass median diameter (MMD), a mass median envelopediameter (MMED) or a mass median geometric diameter (MMGD) of at least 5μm, for example from about 5 μm to about 30 μm.

[0111] Aerodynamically light particles preferably have “mass medianaerodynamic diameter” (MMAD), also referred to herein as “aerodynamicdiameter”, between about 1 μm and about 5 μm. In one embodiment of theinvention, the MMAD is between about 1 μm and about 3 μm. In anotherembodiment, the MMAD is between about 3 μm and about 5 μm.

[0112] In another embodiment of the invention, the particles have anenvelope mass density, also referred to herein as “mass density” of lessthan about 0.4 g/cm³. The envelope mass density of an isotropic particleis defined as the mass of the particle divided by the minimum sphereenvelope volume within which it can be enclosed.

[0113] Tap density can be measured by using instruments known to thoseskilled in the art such as the Dual Platform Microprocessor ControlledTap Density Tester (Vankel, N.C.) or a Geopyc™ instrument (MicrometricsInstrument Corp., Norcross, Ga. 30093). Tap density is a standardmeasure of the envelope mass density. Tap density can be determinedusing the method of USP Bulk Density and Tapped Density, United StatesPharmacopia convention, Rockville, Md., 10^(th) Supplement, 4950-4951,1999. Features which can contribute to low tap density include irregularsurface texture and porous structure.

[0114] The diameter of the particles, for example, their VMGD, can bemeasured using an electrical zone sensing instrument such as aMultisizer IIe, (Coulter Electronic, Luton, Beds, England), or a laserdiffraction instrument (for example Helos, manufactured by Sympatec,Princeton, N.J.). Other instruments for measuring particle diameter arewell known in the art. The diameter of particles in a sample will rangedepending upon factors such as particle composition and methods ofsynthesis. The distribution of size of particles in a sample can beselected to permit optimal deposition within targeted sites within therespiratory tract.

[0115] Experimentally, aerodynamic diameter can be determined byemploying a gravitational settling method, whereby the time for anensemble of particles to settle a certain distance is used to inferdirectly the aerodynamic diameter of the particles. An indirect methodfor measuring the mass median aerodynamic diameter (MMAD) is themulti-stage liquid impinger (MSLI).

[0116] The aerodynamic diameter, d_(aer), can be calculated from theequation:

d _(aer) =d _(g){square root}ρ_(tap)

[0117] where d_(g) is the geometric diameter, for example the MMGD and ρis the powder density.

[0118] Particles which have a tap density less than about 0.4 g/cm³,median diameters of at least about 5 μm, and an aerodynamic diameter ofbetween about 1 μm and about 5 μm, preferably between about 1 μm andabout 3 μm, are more capable of escaping inertial and gravitationaldeposition in the oropharyngeal region, and are targeted to the airwaysor the deep lung. The use of larger, more porous particles isadvantageous since they are able to aerosolize more efficiently thansmaller, denser aerosol particles such as those currently used forinhalation therapies.

[0119] In comparison to smaller particles the larger aerodynamicallylight particles, preferably having a VMGD of at least about 5 μm, alsocan potentially more successfully avoid phagocytic engulfment byalveolar macrophages and clearance from the lungs, due to size exclusionof the particles from the phagocytes' cytosolic space. Phagocytosis ofparticles by alveolar macrophages diminishes precipitously as particlediameter increases beyond about 3 μm. Kawaguchi, H., et al.,Biomaterials, 7: 61-66 (1986); Krenis, L. J. and Strauss, B., Proc. Soc.Exp. Med., 107: 748-750 (1961); and Rudt, S. and Muller, R. H., J.Contr. Rel., 22: 263-272 (1992). For particles of statisticallyisotropic shape, such as spheres with rough surfaces, the particleenvelope volume is approximately equivalent to the volume of cytosolicspace required within a macrophage for complete particle phagocytosis.

[0120] The particles may be fabricated with the appropriate material,surface roughness, diameter and tap density for localized delivery toselected regions of the respiratory tract such as the deep lung or upperor central airways. For example, higher density or larger particles maybe used for upper airway delivery, or a mixture of varying sizedparticles in a sample, provided with the same or different therapeuticagent may be administered to target different regions of the lung in oneadministration. Particles having an aerodynamic diameter ranging fromabout 3 to about 5 μm are preferred for delivery to the central andupper airways. Particles having an aerodynamic diameter ranging fromabout 1 to about 3 μm are preferred for delivery to the deep lung.

[0121] Inertial impaction and gravitational settling of aerosols arepredominant deposition mechanisms in the airways and acini of the lungsduring normal breathing conditions. Edwards, D. A., J. Aerosol Sci., 26:293-317 (1995). The importance of both deposition mechanisms increasesin proportion to the mass of aerosols and not to particle (or envelope)volume. Since the site of aerosol deposition in the lungs is determinedby the mass of the aerosol (at least for particles of mean aerodynamicdiameter greater than approximately 1 μm), diminishing the tap densityby increasing particle surface irregularities and particle porositypermits the delivery of larger particle envelope volumes into the lungs,all other physical parameters being equal.

[0122] The low tap density particles have a small aerodynamic diameterin comparison to the actual envelope sphere diameter. The aerodynamicdiameter, d_(aer), is related to the envelope sphere diameter, d (Gonda,I., “Physico-chemical principles in aerosol delivery,” in Topics inPharmaceutical Sciences 1991 (eds. D. J. A. Crommelin and K. K. Midha),pp. 95-117, Stuttgart: Medpharm Scientific Publishers, 1992)), by theformula:

d _(aer) =d{square root}ρ

[0123] where the envelope mass ρ is in units of g/cm³. Maximaldeposition of monodispersed aerosol particles in the alveolar region ofthe human lung (˜60%) occurs for an aerodynamic diameter ofapproximately d_(aer)=3 μm. Heyder, J. et al., J. Aerosol Sci., 17:811-825 (1986). Due to their small envelope mass density, the actualdiameter d of aerodynamically light particles comprising a monodisperseinhaled powder that will exhibit maximum deep-lung deposition is:

d=3/{square root}ρ μm (where ρ<1 g/cm³);

[0124] where d is always greater than 3 μm. For example, aerodynamicallylight particles that display an envelope mass density, ρ=0.1 g/cm³, willexhibit a maximum deposition for particles having envelope diameters aslarge as 9.5 μm. The increased particle size diminishes interparticleadhesion forces. Visser, J., Powder Technology, 58: 1-10. Thus, largeparticle size increases efficiency of aerosolization to the deep lungfor particles of low envelope mass density, in addition to contributingto lower phagocytic losses.

[0125] The aerodyanamic diameter can be calculated to provide formaximum deposition within the lungs, previously achieved by the use ofvery small particles of less than about five microns in diameter,preferably between about one and about three microns, which are thensubject to phagocytosis. Selection of particles which have a largerdiameter, but which are sufficiently light (hence the characterization“aerodynamically light”), results in an equivalent delivery to thelungs, but the larger size particles are not phagocytosed. Improveddelivery can be obtained by using particles with a rough or unevensurface relative to those with a smooth surface.

[0126] Suitable particles can be fabricated or separated, for example byfiltration or centrifugation, to provide a particle sample with apreselected size distribution. For example, greater than about 30%, 50%,70%, or 80% of the particles in a sample can have a diameter within aselected range of at least about 5 μm. The selected range within which acertain percentage of the particles must fall may be for example,between about 5 and about 30 μm, or optimally between about 5 and about15 μm. In one preferred embodiment, at least a portion of the particleshave a diameter between about 9 and about 11 μm. Optionally, theparticle sample also can be fabricated wherein at least about 90%, oroptionally about 95% or about 99%, have a diameter within the selectedrange. The presence of the higher proportion of the aerodynamicallylight, larger diameter particles in the particle sample enhances thedelivery of therapeutic or diagnostic agents incorporated therein to thedeep lung. Large diameter particles generally mean particles having amedian geometric diameter of at least about 5 μm.

[0127] The particles can be prepared by spray drying. For example, aspray drying mixture, also referred to herein as “feed solution” or“feed mixture”, which includes the bioactive agent and one or morecharged lipids having a charge opposite to that of the active agent uponassociation are fed to a spray dryer.

[0128] For example, when employing a protein active agent, the agent maybe dissolved in a buffer system above or below the pI of the agent.Specifically, insulin for example may be dissolved in an aqueous buffersystem (e.g., citrate, phosphate, acetate, etc.) or in 0.01 N HCl. ThepH of the resultant solution then can be adjusted to a desired valueusing an appropriate base solution (e.g., 1 N NaOH). In one preferredembodiment, the pH may be adjusted to about pH 7.4. At this pH insulinmolecules have a net negative charge (pI=5.5). In another embodiment,the pH may be adjusted to about pH 4.0. At this pH insulin moleculeshave a net positive charge (pI=5.5). Typically the cationic phospholipidis dissolved in an organic solvent or combination of solvents. The twosolutions are then mixed together and the resulting mixture is spraydried.

[0129] For a small molecule active agent, the agent may be dissolved ina buffer system above or below the pKa of the ionizable group(s).Specifically, albuterol sulfate or estrone sulfate, for example, can bedissolved in an aqueous buffer system (e.g., citrate, phosphate,acetate, etc.) or in sterile water for irrigation. The pH of theresultant solution then can be adjusted to a desired value using anappropriate acid or base solution. If the pH is adjusted to about pH 3to about pH 8 range, estrone sulfate will possess one negative chargeper molecule and albuterol sulfate will possess one positive charge permolecule. Therefore, charge interaction can be engineered by the choiceof an appropriate phospholipid. Typically the negatively charged or thepositively charged phospholipid is dissolved in an organic solvent orcombination of solvents and the two solutions are then mixed togetherand the resulting mixture is spray dried.

[0130] Suitable organic solvents that can be present in the mixturebeing spray dried include, but are not limited to, alcohols for example,ethanol, methanol, propanol, isopropanol, butanols, and others. Otherorganic solvents include, but are not limited to, perfluorocarbons,dichloromethane, chloroform, ether, ethyl acetate, methyl tert-butylether and others. Aqueous solvents that can be present in the feedmixture include water and buffered solutions. Both organic and aqueoussolvents can be present in the spray-drying mixture fed to the spraydryer. In one embodiment, an ethanol water solvent is preferred with theethanol:water ratio ranging from about 50:50 to about 90:10. The mixturecan have a, acidic or alkaline pH. Optionally, a pH buffer can beincluded. Preferably, the pH can range from about 3 to about 10.

[0131] The total amount of solvent or solvents being employed in themixture being spray dried generally is greater than 99 weight percent.The amount of solids (drug, charged lipid and other ingredients) presentin the mixture being spray dried generally is less than about 1.0 weightpercent. Preferably, the amount of solids in the mixture being spraydried ranges from about 0.05% to about 0.5% by weight.

[0132] Using a mixture which includes an organic and an aqueous solventin the spray drying process allows for the combination of hydrophilicand hydrophobic components, while not requiring the formation ofliposomes or other structures or complexes to facilitate solubilizationof the combination of such components within the particles.

[0133] Suitable spray-drying techniques are described, for example, byK. Masters in “Spray Drying Handbook”, John Wiley & Sons, New York,1984. Generally, during spray-drying, heat from a hot gas such as heatedair or nitrogen is used to evaporate the solvent from droplets formed byatomizing a continuous liquid feed. Other spray-drying techniques arewell known to those skilled in the art. In a preferred embodiment, arotary atomizer is employed. An example of a suitable spray dryer usingrotary atomization includes the Mobile Minor spray dryer, manufacturedby Niro, Denmark. The hot gas can be, for example, air, nitrogen orargon.

[0134] Preferably, the particles of the invention are obtained by spraydrying using an inlet temperature between about 100° C. and about 400°C. and an outlet temperature between about 50° C. and about 130° C.

[0135] The spray dried particles can be fabricated with a rough surfacetexture to reduce particle agglomeration and improve flowability of thepowder. The spray-dried particle can be fabricated with features whichenhance aerosolization via dry powder inhaler devices, and lead to lowerdeposition in the mouth, throat and inhaler device.

[0136] The particles of the invention can be employed in compositionssuitable for drug delivery via the pulmonary system. For example, suchcompositions can include the particles and a pharmaceutically acceptablecarrier for administration to a patient, preferably for administrationvia inhalation. The particles can be co-delivered with larger carrierparticles, not including a therapeutic agent, the latter possessing massmedian diameters for example in the range between about 50 μm and about100 μm. The particles can be administered alone or in any appropriatepharmaceutically acceptable carrier, such as a liquid, for examplesaline, or a powder, for administration to the respiratory system.

[0137] Particles including a medicament, for example one or more of thedrugs listed above, are administered to the respiratory tract of apatient in need of treatment, prophylaxis or diagnosis. Administrationof particles to the respiratory system can be by means such as known inthe art. For example, particles are delivered from an inhalation device.In a preferred embodiment, particles are administered via a dry powderinhaler (DPI). Metered-dose-inhalers (MDI), nebulizers or instillationtechniques also can be employed.

[0138] Various suitable devices and methods of inhalation which can beused to administer particles to a patient's respiratory tract are knownin the art. For example, suitable inhalers are described in U.S. Pat.No. 4,069,819, issued Aug. 5, 1976 to Valentini, et al., U.S. Pat. No.4,995,385 issued Feb. 26, 1991 to Valentini, et al., and U.S. Pat. No.5,997,848 issued Dec. 7, 1999 to Patton, et al. Various suitable devicesand methods of inhalation which can be used to administer particles to apatient's respiratory tract are known in the art. For example, suitableinhalers are described in U.S. Pat. Nos. 4,995,385, and 4,069,819 issuedto Valentini, et al., U.S. Pat. No. 5,997,848 issued to Patton. Otherexamples include, but are not limited to, the Spinhaler® (Fisons,Loughborough, U.K.), Rotahaler® (Glaxo-Wellcome, Research TriangleTechnology Park, North Carolina), FlowCaps®) (Hovione, Loures,Portugal), Inhalator® (Boehringer-Ingelheim, Germany), and theAerolizer® (Novartis, Switzerland), the diskhaler (Glaxo-Wellcome, RTP,NC) and others, such as known to those skilled in the art. Preferably,the particles are administered as a dry powder via a dry powder inhaler.

[0139] Preferably, particles administered to the respiratory tracttravel through the upper airways (oropharynx and larynx), the lowerairways which include the trachea followed by bifurcations into thebronchi and bronchioli and through the terminal bronchioli which in turndivide into respiratory bronchioli leading then to the ultimaterespiratory zone, the alveoli or the deep lung. In a preferredembodiment of the invention, most of the mass of particles deposits inthe deep lung. In another embodiment of the invention, delivery isprimarily to the central airways. Delivery to the upper airways can alsobe obtained.

[0140] In one embodiment of the invention, delivery to the pulmonarysystem of particles is in a single, breath-actuated step, as describedin U.S. patent application, High Efficient Delivery of a LargeTherapeutic Mass Aerosol, application Ser. No. 09/591,307, filed Jun. 9,2000, which is incorporated herein by reference in its entirety. Inanother embodiment of the invention, at least 50% of the mass of theparticles stored in the inhaler receptacle is delivered to a subject'srespiratory system in a single, breath-activated step. In a furtherembodiment, at least 5 milligrams and preferably at least 10 milligramsof a medicament is delivered by administering, in a single breath, to asubject's respiratory tract particles enclosed in the receptacle.Amounts as high as 15, 20, 25, 30, 35, 40 and 50 milligrams can bedelivered.

[0141] As used herein, the term “effective amount” means the amountneeded to achieve the desired therapeutic or diagnostic effect orefficacy. The actual effective amounts of drug can vary according to thespecific drug or combination thereof being utilized, the particularcomposition formulated, the mode of administration, and the age, weight,condition of the patient, and severity of the symptoms or conditionbeing treated. Dosages for a particular patient can be determined by oneof ordinary skill in the art using conventional considerations, (e.g. bymeans of an appropriate, conventional pharmacological protocol). Forexample, effective amounts of albuterol sulfate range from about 100micrograms (μg) to about 10 milligrams (mg).

[0142] Aerosol dosage, formulations and delivery systems also may beselected for a particular therapeutic application, as described, forexample, in Gonda, I. “Aerosols for delivery of therapeutic anddiagnostic agents to the respiratory tract,” in Critical Reviews inTherapeutic Drug Carrier Systems, 6: 273-313, 1990; and in Moren,“Aerosol dosage forms and formulations,” in: Aerosols in Medicine.Principles, Diagnosis and Therapy, Moren, et al., Eds, Esevier,Amsterdam, 1985.

[0143] Drug release rates can be described in terms of releaseconstants. The first order release constant can be expressed using thefollowing equations:

M _((t)) =M _((∞))*(1−e ^(−k*t))  (1)

[0144] Where k is the first order release constant. M_((∞)) is the totalmass of drug in the drug delivery system, e.g. the dry powder, andM_((t)) is the amount of drug mass released from dry powders at time t.

[0145] Equations (1) may be expressed either in amount (i.e., mass) ofdrug released or concentration of drug released in a specified volume ofrelease medium. For example, Equation (1) may be expressed as:

C _((t)) =C _((∞))*(1−e ^(−k*t)) or Release_((t))=Release_((∞))*(1−e^(−k*t))  (2)

[0146] Where k is the first order release constant. C_((∞)) is themaximum theoretical concentration of drug in the release medium, andC_((t)) is the concentration of drug being released from dry powders tothe release medium at time t.

[0147] Drug release rates in terms of first order release constant canbe calculated using the following equations:

k=−ln(M _((∞)) −M _((t)))/M _((∞)) /t  (3)

[0148] The release constants presented in Tables 4 and 8 employ equation(2).

[0149] As used herein, the term “a” or “an” refers to one or more.

[0150] The term “nominal dose” as used herein, refers to the total massof bioactive agent which is present in the mass of particles targetedfor administration and represents the maximum amount of bioactive agentavailable for administration.

EXEMPLIFICATION

[0151] Materials

[0152] Humulin L (human insulin zinc suspension) was obtained from Lilly(100 U/mL)

[0153] Mass Median Aerodynamic Diameter—MMAD (μm)

[0154] The mass median aerodynamic diameter was determined using anAerosizer/Aerodisperser (Amherst Process Instrument, Amherst, Mass.).Approximately 2 mg of powder formulation was introduced into theAerodisperser and the aerodynamic size was determined by time of flightmeasurements.

[0155] Volume Median Geometric Diameter—VMGD (μm)

[0156] The volume median geometric diameter was measured using a RODOSdry powder disperser (Sympatec, Princeton, N.J.) in conjunction with aHELOS laser diffractometer (Sympatec). Powder was introduced into theRODOS inlet and aerosolized by shear forces generated by a compressedair stream regulated at 2 bar. The aerosol cloud was subsequently drawninto the measuring zone of the HELOS, where it scattered light from alaser beam and produced a fraunhofer diffraction pattern used to inferthe particle size distribution and determine the median value.

[0157] Where noted, the volume median geometric diameter was determinedusing a Coulter Multisizer II. Approximately 5-10 mg powder formulationwas added to 50 mL isoton II solution until the coincidence of particleswas between 5 and 8%.

[0158] Determination of Plasma Insulin Levels

[0159] Quantification of insulin in rat plasma was performed using ahuman insulin specific RIA kit (Linco Research, Inc., St. Charles, Mo.,catalog #HI-14K). The assay shows less than 0.1% cross reactivity withrat insulin. The assay kit procedure was modified to accommodate the lowplasma volumes obtained from rats, and had a sensitivity ofapproximately 5 μU/mL.

[0160] Determination of Estrone-Sulfate Plasma Levels

[0161] Quantification of estrone-sulfate in rat plasma was performedusing an estrone-sulfate RIA kit (Diagnostic Systems Laboratories, Inc.,Webster, Tex., catalog #DSL-C5400). The assay kit procedure was modifiedto accommodate the low plasma volumes obtained from rats and to correctfor influence of the human serum standard matrix, and had a sensitivityof approximately 0.025 ng/mL.

[0162] Preparation of Insulin Formulations

[0163] The powder formulations listed in Table 1 were prepared asfollows. Pre-spray drying solutions were prepared by dissolving thelipid in ethanol and the insulin, leucine, and/or sodium citrate inwater. The ethanol solution was then mixed with the water solution at aratio of 60/40 ethanol water. Final total solute concentration of thesolution used for spray drying varied from 1 g/L to 3 g/L. As anexample, the DPPC/citrate/insulin (60/10/30) spray drying solution wasprepared by dissolving 600 mg DPPC in 600 mL of ethanol, dissolving 100mg of sodium citrate and 300 mg of insulin in 400 mL of water and thenmixing the two solutions to yield one liter of cosolvent with a totalsolute concentration of 1 g/L (w/v). Higher solute concentrations of 3g/L (w/v) were prepared by dissolving three times more of each solute inthe same volumes of ethanol and water.

[0164] The solution was then used to produce dry powders. A NitroAtomizer Portable Spray Dryer (Niro, Inc., Columbus, Md.) was used.Compressed air with variable pressure (1 to 5 bar) ran a rotary atomizer(2,000 to 30,000 rpm) located above the dryer. Liquid feed with varyingrate (20 to 66 mL/min) was pumped continuously by an electronic meteringpump (LMI, Model #Al 51-192s) to the atomizer. Both the inlet and outlettemperatures were measured. The inlet temperature was controlledmanually; it could be varied between 100° C. and 400° C. and wasestablished at 100, 110, 150, 175 or 200° C., with a limit of control of5° C. The outlet temperature was determined by the inlet temperature andsuch factors as the gas and liquid feed rates (it varied between 50° C.and 130° C.). A container was tightly attached to the cyclone forcollecting the powder product. TABLE 1 POWDER FORMU- COMPOSITION (%)LATION Ci- In- NUMBER DPePC DSePC DPPG DPPC Leucine trate sulin  1† 7010 20  2 70 20 10  3 70 10 20  4 50 50  5‡ 40 10 50  6 70 10 20  7 50 50 8 54.5 45.5  9 50 10 40 10 70 10 2 11 70 8 2 20 12† 40 10 50 13† 60 1030 13A† 60 10 30 14‡ 70 20 15† 70 20 10

[0165] The physical characteristic of the insulin containing powders isset forth in Table 2. The MMAD and VMGD were determined as detailedabove. TABLE 2 COMPOSITIONS MMAD VMGD Density Formulations (% WEIGHTBASIS) (μm)§ (μm)¶ (g/cc)‡ Humulin R — — — — Humulin L — — — — Humulin U— — — —  1 DPPC/Leu/Insulin (Sigma) = 70/10/20 2.6 13.4 0.038  2 DSePC(Avanti)/Leu/Insulin (Sigma) = 70/10/20 3.3 10.0 0.109  3 DSePC(Avanti)/Leu/Insulin (Sigma) = 70/10/20 3.4 13.6 0.063  4 DPePC(Avanti)/Insulin (Sigma) = 50/50 3.2 15.3 0.044  5 DPPG/SodiumCitrate/Insulin = 40/10/50 3.9 11.6 0.113  6 DPePC (Genzyme)/Leu/Insulin(BioBras) = 70/10/20 2.6 9.1 0.082  7 DPePC (Avanti)/Insulin (BioBras) =50/50 2.8 11.4 0.060  8 DPePC (Genzyme)/Insulin (BioBras) = 54.5/45.52.8 12.6 0.049  9 DPePC (Genzyme)/Leu/Insulin (BioBras) = 50/10/40 2.28.4 0.069 10 DPePC (Avanti)/Leu/Insulin (BioBras) = 70/10/20 3.7 15.50.057 11 DPePC (Avanti)/Leu/Sodium Citrate/Insulin 2.6 15.3 0.029(BioBras) = 70/8/2/20 12 DPPC/Sodium Citrate/Insulin = 40/10/50 3.5 11.60.091 13 DPPC/Insulin/Sodium Citrate = 60/30/10 1.9 8.0 0.056

[0166] The data presented in Table 2 showing the physicalcharacteristics of the formulations comprising insulin are predictive ofthe respirability of the formulations. That is, as discussed above thelarge geometric diameters, small aerodynamic diameters and low densitiespossessed by the powder prepared as described herein render theparticles respirable.

[0167] In Vivo Insulin Experiments

[0168] The following experiment was performed to determine the rate andextent of insulin absorption into the blood stream of rats followingpulmonary administration of dry powder formulations comprising insulinto rats.

[0169] The nominal insulin dose administered was 100 μg per rat. Toachieve the nominal doses, the total weight of powder administered perrat ranged from 0.2 mg to 1 mg, depending on percent composition of eachpowder. Male Sprague-Dawley rats were obtained from Taconic Farms(Germantown, N.Y.). At the time of use, the animals weighed 386 g inaverage (±5 g S.E.M.). The animals were allowed free access to food andwater.

[0170] The powders were delivered to the lungs using an insufflatordevice for rats (PennCentury, Philadelphia, Pa.). The powder amount wastransferred into the insufflator sample chamber. The delivery tube ofthe insufflator was then inserted through the mouth into the trachea andadvanced until the tip of the tube was about a centimeter from thecarina (first bifurcation). The volume of air used to deliver the powderfrom the insufflator sample chamber was 3 mL, delivered from a 10 mLsyringe. In order to maximize powder delivery to the rat, the syringewas recharged and discharged two more times for a total of three airdischarges per powder dose.

[0171] The injectable insulin formulation Humulin L was administered viasubcutaneous injection, with an injection volume of 7.2 μL for a nominaldose of 25 μg insulin; Catheters were placed into the jugular veins ofthe rats the day prior to dosing. At sampling times, blood samples weredrawn from the jugular vein catheters and immediately transferred toEDTA coated tubes. Sampling times were 0, 0.25, 0.5, 1, 2, 4, 6, 8, and24 hrs. after powder administration. In some cases an additionalsampling time (12 hrs.) was included, and/or the 24 hr. time pointomitted. After centrifugation, plasma was collected from the bloodsamples. Plasma samples were stored at 4° C. if analysis was performedwithin 24 hours or at −75° C. if analysis would occur later than 24hours after collection. The plasma insulin concentration was determinedas described above.

[0172] Table 3 contains the insulin plasma levels quantified using theassay described above. TABLE 3 PLASMA INSULIN CONCENTRATION (μU/mL) ±S.E.M. Time Hum- (hrs) 1 2 3 4 5 6 13A 14 lin L 15 0   5.0 ±  5.2 ±  5.0±  5.0 ±  5.3 ±  5.7 ±   5.0 ±  5.0 ±  5.0 ±   5.0 ±   0.0  0.2  0.0 0.0  0.2  0.7   0.0  0.0  0.0   0.0 0.25 1256.4 ± 61.6 ±  98.5 ± 518.2± 240.8 ± 206.8 ± 1097.7 ± 933.9 ± 269.1 ± 1101.9 ±  144.3 22.5  25.3179.2  67.6  35.1  247.5 259.7  82.8  258.9 0.5 1335.8 ± 85.2 ± 136.7 ±516.8 ± 326.2 ± 177.3 ±  893.5 ± 544.9 ± 459.9 ± 1005.4 ±  81.9 21.7 37.6 190.9 166.9  7.8  177.0 221.1  91.6  263.9 1  859.0 ± 85.4 ± 173.0± 497.0 ± 157.3 ± 170.5 ±  582.5 ± 229.6 ± 764.7 ±  387.5 ±  199.4 17.6 28.8  93.9  52.5  32.9  286.3  74.4 178.8 1143.9 2  648.6 ± 94.8 ±158.3 ± 496.5 ± 167.7 ± 182.2 ±  208.5 ± 129.8 ± 204.4 ±  343.8 ±  171.125.0  39.1 104.9  70.5  75.0  78.3  45.7  36.7  95.3 4  277.6 ± 52.5 ± 98.0 ± 343.8 ± 144.8 ± 170.2 ±  34.9 ±  41.9 ±  32.1 ±  170.6 ±  86.8 9.1  24.3  66.7  43.8  56.3   5.4  28.7  22.6  79.9 6  104.0 ± 33.0 ± 58.7 ± 251.2 ±  95.7 ± 159.5 ±  12.3 ±  9.0 ±  11.1 ±  15.4 ±  43.110.7  4.1  68.4  27.3  43.4   2.4  2.9  7.5   4.5 8  54.4 ± 30.2 ±  42.5±  63.2 ±  52.5 ±  94.8 ±   5.2 ±  5.0 ±  5.5 ±   6.5 ±  34.7  8.1  17.8 16.5  13.7  23.5   0.1  0.0  2.1   0.6 12  17.2 ±  6.5 24  5.0 ±  5.5 ± 0.0  0.3 n   5  5  6  6  6  6  8

[0173] The in vivo release data of Table 3 show that powder formulationscomprising insulin and positively charged lipids (DPePC and DSePC) havesignificantly lower initial burst of insulin than that seen with powderformulations comprising insulin and the lipid DPPC (Formulations 1 and13) and sustained elevated levels at 6 to 8 hours. FIG. 1 sets forth therelease profile for insulin from Formulations 2, 3, 6 and 15.

[0174] In addition, the use of charged lipids having a charge which isthe same of the active at neutral pH, can also be employed provided thatthe preparation of the spray dried formulation is conducted at a pHwhere the lipid and active agent possess overall charges which areopposite and are therefore capable of charge interaction. See, forexample, Formulations 5 which employs the negatively charged lipid DPPG.Formulation 5 was prepared and spray dried at a pH of about 4.0. At thispH, DPPG is negatively charged and insulin becomes positively charged(pI=5.5) thereby providing for a charge interaction to occur. However,when the DPPG and insulin are prepared and spray dried at pH=7.4 whereboth the DPPG and insulin possess an overall negative charge,Formulation 14, the proper environment for charge interaction to occuris not provided. It is noted that Formulation 5 showed a significantlylower initial burst of insulin (240.8±67.6 μU/mL) as compared toFormulation 14 (933.9±259.7 μU/mL) with higher sustained levels at 6 to8 hours post treatment. FIG. 2 shows a comparison of the in vivo releaseprofile for Formulations 5, 14 and 13A (lipid, DPPC).

[0175] In Vitro Analysis of Insulin-Containing Formulations

[0176] The in vitro release of insulin containing dry powderformulations was performed as described by Gietz et al. in Eur. J.Pharm. Biopharm., 45:259-264 (1998), with several modifications.Briefly, in 20 mL screw-capped glass scintillation vials about 10 mg ofeach dry powder formulation was mixed with 4 mL of warm (37° C.) 1%agarose solution using polystyrene stir bars. The resulting mixture wasthen distributed in 1 mL aliquots to a set of five fresh 20 mL glassscintillation vials. The dispersion of dry powder in agarose was cooledin an ambient temperature dessicator box protected from light to allowgelling. Release studies were conducted on an orbital shaker at about37° C. At predetermined time points, previous release medium (1.5 mL)was removed and fresh release medium (1.5 mL) was added to each vial.Typical time points for these studies were 5 minutes, 1, 2, 4, 6 and 24hours. The release medium used consisted of 20 mM4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES), 138 mMNaCl, 0.5% Pluronic (Synperonic PE/F68; to prevent insulin filbrillationin the release medium); pH 7.4. A Pierce (Rockford, Ill.) protein assaykit (See Anal Biochem, 150:76-85 (1985)) using known concentrations ofinsulin standard was used to monitor insulin concentrations in therelease medium.

[0177] Table 4 summarizes the in vitro release data and first orderrelease constants for powder formulations of Table 1 comprising insulin.TABLE 4 Powder Cumulative % Cumulative % Maximum ‡ Release First Order ‡Formulation Insulin Released Insulin Released at 24 hr Release Number at6 hr at 24 hr (Cumulative %) Constants (hr⁻¹) Humulin R 92.67 ± 0.3694.88 ± 0.22  91.6 ± 5.42 1.0105 ± 0.2602 Humulin L 19.43 ± 0.41 29.71 ±0.28  36.7 ± 2.56 0.0924 ± 0.0183 Humulin U  5.17 ± 0.18 12.65 ± 0.43 46.6 ± 27.0 0.0158 ± 0.0127  2 31.50 ± 0.33 47.52 ± 0.43 48.22 ± 0.460.1749 ± 0.0038  3 26.34 ± 0.71 37.49 ± 0.27 38.08 ± 0.72 0.1837 ±0.0079  4 24.66 ± 0.20 31.58 ± 0.33 31.51 ± 1.14 0.2457 ± 0.0214  529.75 ± 0.17 35.28 ± 0.19 33.66 ± 2.48 0.4130 ± 0.0878  6 17.04 ± 0.7124.71 ± 0.81 25.19 ± 0.52 0.1767 ± 0.0083  7 13.53 ± 0.19 19.12 ± 0.4019.51 ± 0.48 0.1788 ± 0.0101  8 13.97 ± 0.27 17.81 ± 0.46 17.84 ± 0.550.2419 ± 0.0178  9 17.47 ± 0.38 22.17 ± 0.22 21.97 ± 0.64 0.2734 ±0.0196 10 25.96 ± 0.31 34.94 ± 0.31 35.43 ± 0.90 0.2051 ± 0.0120 1134.33 ± 0.51 47.21 ± 0.47 47.81 ± 0.85 0.1994 ± 0.0082 12 61.78 ± 0.3368.56 ± 0.23 65.20 ± 3.34 0.5759 ± 0.0988 13 78.47 ± 0.40 85.75 ± 0.63 84.9 ± 3.81 0.5232 ± 0.0861

[0178] The data presented in Table 4 show that for insulin containingpowder formulations employing the positively charged lipid DPePC(Formulations 4 and 6-11) and DSePC (Formulations 2 and 3), first orderrelease constants similar to that observed with the slow releaseinjectable insulin formulation, Humulin L, can be achieved. Further, thefirst order release constants of these same formulations issignificantly lower than that observed with the fast release injectableinsulin formulation, Humulin R. As such, sustained release dry powderinsulin formulations having varying compositions of positively chargedlipid can be formulated.

[0179] Preparation of Estrone Sulfate-Containing Powder Formulations

[0180] The estrone sulfate powder formulations listed in Table wereprepared as follows. Pre-spray drying solutions were prepared bydissolving the lipin in ethanol and estrone sulfate and leucine inwater. The ethanol solution was then mixed with the water solution at aration 70/30 ethanol/water. Final total solute concentration of thesolution used for spray drying varied from 1 g/L to 3 g/L. As anexample, the DPePC/leucine/estrone sulfate (76/20/4) spray dryingsolution was prepared by dissolving 760 mg of DPePC in 700 mL ofethanol, dissolving 200 mg of leucine and 40 mg of estrone sulfate in300 mL of water and then mixing the two solutions to yield one liter ofcosolvent with a total solute concentration of 1 g/L (w/v). Highersolute concentrations of, for example, 3 g/L (w/) were prepared bydissolving three times more of each solute in the same volumes ofethanol and water

[0181] The mixture was spray dried following the procedure describedabove for the insulin containing powder formulation. During spraydrying, the feed rate was about 50 mL/min, the inlet temperature rangedfrom about 110° C. to about 120° C., and the outlet temperature wasabout 52° C.

[0182] The physical characteristic of the estrone sulfate containingpowders is set forth in Table 5. The MMAD and VMGD were determined asdetailed above. TABLE 5 POWDER FORMULA- COMPOSITIONS TION (% WEIGHT MMADVMGD DENSITY NUMBER BASIS) (μm) § (μm) ¶ (g/cc) ‡ 16 DPePC 5.9 16.00.136 (Avanti)/Leucine/ Estrone Sulfate (sodium salt) = 76/20/4 17DPPC/Leucine/ 3.7 12.7 # 0.085 Estrone Sulfate (sodium salt) = 76/20/4

[0183] The data presented in Table 5 showing the physicalcharacteristics of the formulations comprising estrone sulfate arepredictive of the respirability of the formulations. That is, asdiscussed above the large geometric diameters, small aerodynamicdiameters and low densities possessed by the powder prepared asdescribed herein render the particles respirable.

[0184] In Vivo Experiments—Estrone Sulfate Containing Powders

[0185] The following experiment was performed to determine the rate andextent of estrone sulfate absorption into the blood stream of ratsfollowing pulmonary administration of dry powder formulations comprisingestrone sulfate.

[0186] The nominal estrone-sulfate dose administered was 40 μg per rat,in 1 mg of powder. Male Sprague-Dawley rats were obtained from TaconicFarms (Germantown, N.Y.). At the time of use, the animals weighed anaverage of 415 g (±10 g S.E.M.). The animals were allowed free access tofood and water.

[0187] The powders were delivered to the lungs using an insufflatordevice for rats (PennCentury, Philadelphia, Pa.). The powder amount wastransferred into the insufflator sample chamber. The delivery tube ofthe insufflator was then inserted through the mouth into the trachea andadvanced until the tip of the tube was about a centimeter from thecarina (first bifurcation). The volume of air used to deliver the powderfrom the insufflator sample chamber was 3 mL, delivered from a 10 mLsyringe. In order to maximize powder delivery to the rat, the syringewas recharged and discharged two more times for a total of three airdischarges per powder dose.

[0188] Catheters were placed into the jugular veins of the rats the dayprior to dosing. At sampling times, blood samples were drawn from thejugular vein catheters and immediately transferred to EDTA coated tubes.Sampling times were 0, 0.25, 0.5, 1, 2, 4, and 6 hours after powderadministration. After centrifugation, plasma was collected from theblood samples. Plasma samples were stored at 4° C. if analysis wasperformed within 24 hours or at −75° C. if analysis would occur laterthan 24 hours after collection.

[0189] Table 6 contains the estrone sulfate plasma levels quantifiedusing the assay described above. TABLE 6 PLASMA ESTRONE-SULFATECONCENTRATION (ng/mL) ± S.E.M. TIME (HRS) FORMULATION 16 FORMULATION 170 0.07 ± 0.02 0.08 ± 0.05 0.25 12.07 ± 1.96  22.26 ± 8.96  0.5 18.88 ±2.21  23.39 ± 12.72 1 12.20 ± 3.31  10.59 ± 0.61  2 4.65 ± 0.77 3.45 ±0.63 4 4.02 ± 1.42 0.86 ± 0.10 6 1.49 ± 0.48 0.33 ± 0.12 n 4 3

[0190] The results presented in Table 6 and depicted graphically in FIG.3, show that the formulation comprising DPePC (overall positive charge)and estrone sulfate (negative charge) exhibited sustained release ofestrone sulfate when compared to the formulation employing the lipidDPPC (no overall net charge) and estrone sulfate. Specifically, at sixhours post administration, the plasma level of estrone sulfate for theDPePC containing formulation was 1.49±0.48 ng/mL as compared to0.33±0.12 ng/mL for the DPPC containing formulation.

[0191] Preparation of Albuterol-Containing Powder Formulations

[0192] The albuterol sulfate powder formulations listed in Table 7, wereprepared as follows. Pre-spray drying solutions were prepared bydissolving the lipid in ethanol and albuterol sulfate and leucine inwater. The ethanol solution was then mixed with the water solution at aratio of 70/30 ethanol/water. Final total solute concentration of thesolution used for spray drying varied from 1 g/L to 3 g/L. As anexample, the DPPC/leucine/albuterol sulfate (76/16/8) spray dryingsolution was prepared by dissolving 760 mg of DPPC in 700 mL of ethanol,dissolving 160 mg leucine and 870 mg of albuterol sulfate in 300 mLwater and then mixing the two solutions to yield one liter of cosolventwith a total solute concentration of 1 g/L (w/v). Higher soluteconcentrations of 3 g/L (w/v) were prepared by dissolving three timesmore of each solute in the same volumes of ethnaol and water. Thesolution was spray-dried as described above for the insulin containingformulation. Specifically, the inlet temperature was from about 110° C.to about 140° C., and the outlet temperature ranged from about 45-57° C.

[0193] The physical characteristics of the albuterol sulfate containingpowders is set forth in Table 7. The MMAD and VMGD were determined asdetailed above. TABLE 7 POWDER FORMULA- TION COMPOSITIONS MMAD VMGDDENSITY NUMBER (% WEIGHT BASIS) (μm) § (μm) ¶ (g/cc) ‡ 18 DSPC/Leucine/3.3 6.1 0.293 Albuterol Sulfate = 76/16/8 19 DSPG/Leucine/ 4.1 6.4 0.410Albuterol Sulfate = 76/16/8 20 DPPC/Leucine/ 2.8 12.0 0.054 AlbuterolSulfate = 76/23/1 21 DPPG/Leucine/ 3.3 7.1 0.216 Albuterol Sulfate =76/16/8

[0194] The data presented in Table 7 showing the physicalcharacteristics of the formulations comprising albuterol sulfate arepredictive-of the respirability of the formulations. That is, asdiscussed above the large geometric diameters, small aerodynamicdiameters and low densities possessed by the formulations prepared asdescribed herein render the formulations respirable.

[0195] In Vivo Testing of Albuterol Sulfate Formulations

[0196] A whole-body plethysmography method for evaluating pulmonaryfunction in guinea pigs was used to assess the sustained effects of thealbuterol sulfate formulations listed in Table 7.

[0197] The system used was the BUXCO whole-body unrestrainedplethysmograph system with BUXCO XA pulmonary function software (BUXCOElectronics, Inc., Sharon, Conn.). The method was conducted as describedby Silbaugh S. A. and Mauderly, J. L., in American PhysiologicalSociety, Vol. 84:1666-1669 (1984) and Chang, B. T., et al in Journal ofPharmacological and Toxicological Methods, Vol. 39(3)-163-168 (1998).This method allows individual animals to be challenged repeatedly overtime with methacholine given by nebulization. A calculated measurementof airway resistance based on flow parameters, the enhanced pause PenHwas used as a marker for protection from methacholine-inducedbronchoconstriction. Baseline pulmonary function (airwayhyperresponsiveness) values were measured prior to any experimentaltreatment. Airway hyperresponsiveness was then assessed in response tosaline and methacholine at various timepoints (2-3, 16 and 24 hours)following administration of albuterol-sulfate formulations. Average PenHis calculated from data collected between 4 and 9 minutes followingchallenge with saline or methacholine. The percent of baseline PenH ateach timepoint is calculated for each experimental animal. Values fromanimals that received the same albuterol sulfate formulation weresubsequently averaged to determine the mean group response (±standarderror) at each timepoint.

[0198] The nominal dose of albuterol-sulfate administered was 50 μg forthe DPPG-based formulation (#21) and 25 μg for the DPPC-basedformulation (#20). To achieve those nominal doses, the total weights ofpowder administered were 0.625 mg and 2.5 mg, respectively.

[0199] Male Hartley guinea pigs were obtained from Elm Hill BreedingLabs (Chelmsford, Mass.). At the time of use, the animals weighed anaverage of 363 g (±5 g S.E.M.). The animals were allowed free access tofood and water. The powder amount was transferred into the insufflatorsample chamber (insufflation device for guinea pigs, Penn Century,Philadelphia, Pa.). The delivery tube of the insufflator was insertedthrough the mouth into the trachea and advanced until the tip of thetube was about a centimeter from the carina (first bifurcation). Thevolume of the air used to deliver the powder from the insufflator samplechamber was 3 mL, delivered from a 10 mL syringe. In order to maximizepowder delivery to the guinea pig, the syringe was recharged anddischarged two more times for a total of three air discharges per powderdose. Methacholine challenges were performed at time points 2-3, 16 and24 hours after administration.

[0200]FIG. 4 shows that the formulation comprising DPPG (overal negativecharge) and albuterol sulfate (overall positive charge) providedsustained protection against methacholine-induced bronchoconstrictionwhen compared to the formulation comprising DPPC (no overall net charge)and albuterol sulfate for at least 24 hours following administration.

[0201] In another experiment, as much as 200 μg of albuterol sulfate ina DPPC-based formulation did not provide prolonged protection againstinduced bronchoconstriction.

[0202] In Vitro Release Studies—Albuterol Sulfate

[0203] Controlled Release Studies of Albuterol Sulfate were conductedusing the COSTAR™ Brand Transwell Inserts, With Plates, Sterile. Theplates were equipped with 6 wells having an area of 4.7 cm². The insertsize was 24 mm, the pore size was 3.0 μm. A predetermined amount of thepowder to be tested (approximately 10-15 mg) was placed into a HPMC Size#2 capsule. The capsule was then placed inside an inhaler and the powderwas sprayed on the Transwell insert using an in-house vacuum system.Formulations were run in triplicate.

[0204] After spraying, the insert was placed inside the Transwell platecontaining a volume of 1.8 mL of Phosphate Buffered Saline (pH=7.4)which had previously been equilibrated at 37° C. for 30 minutes. TheTranswell plate was hermetically sealed in order to prevent evaporationof the buffer during the experiment.

[0205] The Transwell Experiment was carried out in an incubator at 37°C. on an orbital shaker at a speed of 100 min⁻¹. At specifiedtime-points throughout the experiment, 1.8 mL of phosphate bufferedsaline was removed from the Transwell plate. The inserts were thenplaced into a new Transwell plate containing 1.8 mL of fresh phosphatebuffered saline. Typical Transwell experiments are conducted for 4hours. Samples are withdrawn after 5 min., 15, min., 30, min., 1 h, 1.5h, 2 h, 3 h, and 4 h.

[0206] The amount of albuterol sulfate in the PBS buffer sampled atpredetermined in vitro release time points was quantitated using aRP-HPLC method with Phenomenex Luna 5μ, C8(2), 250×4.6 mm column(Torrance, Calif.) and UV detection at 275 nm.

[0207] Table 8 summarizes the in vitro release data and first orderrelease constants for the powder formulations of Table 7 comprisingalbuterol sulfate. The first order release constants for the powderformulation comprising DSPG (negatively charged) and albuterol sulfateis about 4 time slower compared to the powder formulation comprisingDSPC (no net overall charge) and albuterol sulfate (positive). TABLE 8First Cumulative % Maximum Order Powder Insulin Release ReleaseFormulation Compositions Released at 4 hr Constants Number (% weightbasis) at 4 hr (Cumulative %) ‡ (hr⁻¹) ‡ 18 DSPC/Leucine/Albuterol106.21 ± 1.73 105.64 ± 0.20  29.7360 ± 0.7504 Sulfate (sodium salt) =76/16/8 19 DSPG/Leucine/Albuterol  97.44 ± 0.68  95.13 ± 1.39   7.9334 ±0.6877 Sulfate (sodium salt) = 76/16/8

[0208] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A method for delivery via the pulmonary systemcomprising: administering to the respiratory tract of a patient in needof treatment, prophylaxis or diagnosis an effective amount of particlescomprising: a bioactive agent in association with a charged lipidwherein the charged lipid has an overall net charge which is opposite tothe overall net charge of the agent upon association and wherein releaseof the agent is sustained.
 2. The method of claim 1, wherein associationof the agent and charged lipid comprises an ionic complexation.
 3. Themethod of claim 2, wherein association of the lipid and agent furthercomprises hydrogen bonding.
 4. The method of claim 1, wherein the chargeratio of lipid to bioactive agent is from about 0.25:1 to about 1:0.25.5. The method of claim 4, wherein the charge ratio of lipid to bioactiveagent is from about 0.5:1 to about 1:0.5.
 6. The method of claim 5,wherein the charge ratio of lipid to bioactive agent is about 1:1. 7.The method of claim 1, wherein the bioactive agent is a protein.
 8. Themethod of claim 7, wherein the protein is insulin.
 9. The method ofclaim 8, wherein the sustained release is at least about 6 hours postadministration.
 10. The method of claim 1, wherein the bioactive agentis estrone sulfate.
 11. The method of claim 1, wherein the bioactiveagent is albuterol sulfate.
 12. The method of claim 1, wherein the lipidpossesses and overall net negative charge.
 13. The method of claim 12,wherein the lipid is a1,2-diacyl-sn-glycero-3-[phospho-rac-(1-glycerol)] and a1,2-diacyl-sn-glycerol-3-phosphate.
 14. The method of claim 13, whereinthe 1,2-diacyl-sn-glycero-3-[phospho-rac-(1-glycerol)] lipid isrepresented by Formula I:

wherein, R₁ and R₂ are independently an aliphatic group having fromabout 3 to about 24 carbons.
 15. The method of claim 13, wherein the1,2-diacyl-sn-glycero-3-[phospho-rac-(1-glycerol)] lipid is1,2-distearoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DSPG),1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG),1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol)] (DPPG),1,2-dilauroyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DLPG),1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG) or anycombination thereof.
 16. The method of claim 13, wherein the1,2-diacyl-sn-glycerol-3-phosphate is represented by the Formula II:

wherein, R₁ and R₂ are independently an aliphatic group having fromabout 3 to about 24 carbons.
 17. The method of claim 13, wherein the1,2-diacyl-sn-glycerol-3-phosphate lipid is1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA),1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA),1,2-dilauroyl-sn-glycero-3-phosphate (DLPA),1,2-dioleoyl-sn-glycero-3-phosphate (DOPA),1,2-distearoyl-sn-glycero-3-phosphate (DSPA) or any combination thereof.18. The method of claim 1, wherein the particles have a tap density lessthan about 0.4 g/cm³.
 19. The method of claim 18, wherein the particleshave a tap density less than about 0.1 g/cm³.
 20. The method of claim 1,wherein the particles have a median geometric diameter of from about 5micrometers and about 30 micrometers.
 21. The method of claim 1, whereinthe particles have an aerodynamic diameter of from about 1 to about 5microns.
 22. The method of claim 21, wherein the particles have anaerodynamic diameter of from about 1 to about 3 microns.
 23. The methodof claim 22, wherein the particles have an aerodynamic diameter of fromabout 3 to about 5 microns.
 24. The method of claim 1, wherein deliveryto the pulmonary system includes delivery to the deep lung.
 25. Themethod of claim 1, wherein delivery to the pulmonary system includesdelivery to the central airways.
 26. The method of claim 1, whereindelivery to the pulmonary system includes delivery to the upper airways.27. The method of claim 1, wherein the particles further comprise alipid having no overall net charge.
 28. The method of claim 1, whereinthe particles further comprise a carboxylic acid or salt thereof. 29.The method of claim 28, wherein the carboxylic acid includes at leasttwo carboxyl groups.
 30. The method of claim 1, wherein the particlesfurther comprise a multivalent metal salt or ionic components thereof.31. The method of claim 30, wherein the multivalent salt is a salt of analkaline earth metal.
 32. The method of claim 1, wherein the particlesfurther comprise an amino acid.
 33. The method of claim 32, wherein theamino acid is hydrophobic.
 34. The method of claim 33, wherein thehydrophobic amino acid is leucine, isoleucine, alanine, valine,phenylalanine or any combination thereof.